Robust discrimination between self and non-self molecules is central to the immune system of all organisms. In vertebrate adaptive immune system, self vs. non-self discrimination relies on a series of positive and negative selections of immune receptors (e.g. T cell and B cell receptors). In establishing immunological tolerance against self molecules in T cells, a transcriptional regulator, Aire, plays a central role. Aire up- regulates the expression of thousands of peripheral tissue antigens in medullary thymic epithelial cells, allowing self-reactive T cells to undergo clonal deletion or to develop into regulatory T cells. Despite its central role in T cell development, molecular mechanism of Aire remains poorly understood. Previous studies showed that C-terminal tail (CTT) of Aire (AireCTT) plays a key role in the transcriptional activation of Aire target genes. In our effort to dissect how AireCTT functions as a transcriptional activation domain, we recently found that the AireCTT directly binds Cyclin C (CycC), a component of the CDK8 kinase module of Mediator, and that CycC is important for Aire's transcriptional activity. Mediator is a large, multi-subunit transcriptional co-activator that is densely loaded at super-enhancers and can regulate a broad spectrum of genes. Intriguingly, previous studies suggest that Aire is localized at super-enhancers, altering global chromatin landscape and mediating the target gene activation. We hypothesize that Aire utilizes CTT to bind Mediator and cluster at super-enhancers. Furthermore, Aire is known to be phosphorylated at multiple sites, some of which match the consensus sequence for CDK8 substrates. This also raises the question whether CycC binding plays an additional role of recruiting CDK8 for phosphorylation of Aire. To test these hypotheses and to further define the AireCTT:CycC interaction, we here propose two specific aims. First, we will determine the structural basis for the interaction between AireCTT and CycC, using a combination of crystallography, biochemistry and cell-based assays (Aim 1). Second, we will define the functional consequence of the AireCTT:CycC interaction, with a focus on testing its potential role in super- enhancer localization and post-translational modifications of Aire (Aim 2). We expect that the proposed work would provide key missing links in our understanding of Aire. Furthermore, they would also have a broad impact on the field of transcription in general. This is because our work is poised to identify previously unrecognized functions and mechanisms of the CDK8 kinase module, the poorly understood part of Mediator.